JP2006081542A - Method for creating clone mammal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for creating a clone mammal efficiently in a high survival ratio, to provide the clone mammal obtained by the method, to provide a descendant of the same, to provide an embryo of the same, to provide a method for obtaining an ES (embryonic stem) cell from the embryo of the clone mammal, and to provide the ES cell obtained by the method. <P>SOLUTION: This method for creating the clone mammal comprises using a natural killer T cell of a mammal as a donor cell. The clone mammal obtained by the method, the method for obtaining the ES cell from the embryo of the clone mammal, and the ES cell obtained by the method are provided, respectively. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to cloned mammalian embryos and cloned mammals, methods for producing the same, uses thereof, and the like. Furthermore, the present invention relates to a cloned mammalian embryonic stem cell (hereinafter also referred to as ES cell), a method for producing the same, their use, and the like.

Since the birth of cloned sheep Dolly in the UK (see Non-Patent Document 1), animal clones have been established by various techniques, but the somatic cell-derived nuclei are directly transplanted into the nucleated egg to obtain a clone Was very inefficient, and it was very difficult to identify the origin of the donor cells used for transplantation, and there was a problem in reproducibility. To overcome the latter, lymphocytes expressing cell-specific surface markers were used as donors, but they were inefficient and required extra processes. For example, when peripheral lymphocytes such as T cells and B cells are used, ES cells are established only with a probability of about 1/500 (0.2%) (see Non-Patent Document 2). In addition, when the neuroolfactory cells are used as donors, the probability of ES cell establishment is somewhat increased. In any of these cases, the two steps of ES cell establishment and tetraploid complementation are indispensable for clone individual production (non- (See Patent Document 3). Thus, it was very time consuming and laborious to create a clone using peripheral terminally differentiated cells. On the other hand, when considering application to humans, once passing through ES cells, the HLA of the clone does not match that of the donor (except in the case of autotransplantation), which inevitably causes immune rejection and is not used. Is possible. Therefore, identification of somatic cells that can be used as donor cells that surpass these technical and immunological constraints has been demanded. In addition, it has been almost impossible to accurately identify the origin of donor cells by conventional somatic cell cloning techniques, and it has been impossible to specify a cell group capable of regenerating all individuals.

In addition, in the conventional cloning technique, after the embryo transfer, the number of the pups actually reaching the offspring is very small, and there is a demand for a method for producing a cloned mammal that has a higher probability of reaching the offspring.
Wilmut, I .; Et al., “Viable offspring derived from fetal and adult mammarian cells.”, Nature, (UK), 1997, 385, pp. 810-813. Hochedlinger, K.M. , And R.M. Jaenisch. , “Monoclonal microphone generated by nuclear transfer form B and T donor cells.”, Nature, (UK), 2002, 415, p. 1035-1038 Eggan, K .; Et al., “Mice Cloned from Facility Science Neurons.”, Nature, (UK), 2004, 428, p. 44-49 Hwang, W.H. S. Et al., “Evidence of a private human embronic stem cell line derived from a blasted blast system”, Science, (USA), 2004, 303, p. 1669-1674 Wakayama, T .; Et al., “Mic Cloned from Embryonic Stem cells.”, Proceedings of the National Academy of Sciences of United States of America, 1996, USA. 14984-14989

  The present invention provides a method for producing a cloned animal that is more efficient and has a high survival rate, a cloned animal obtained by the method, its progeny, an embryo, and the like, and cells, tissues, organs, products, and the like obtained therefrom. With the goal. Another object of the present invention is to provide a method of obtaining ES cells from a cloned animal embryo, a cloned animal ES cell obtained by the method, and a cell, tissue, organ, product and the like obtained therefrom. Yet another object of the present invention is to provide a somatic cell cloning technique that can accurately identify the origin of donor cells.

  As a result of intensive studies in view of the above-mentioned problems, the present inventors have found that cloned embryos obtained by using natural killer T cells (hereinafter also referred to as NKT cells) as donor cells that provide nuclei are ES cells. It has been found that the development and the littering can be achieved without the need for the establishment of and the sequential tetracomplementation. Furthermore, it was confirmed that the obtained cloned animal had normal fertility, and the present invention was completed. That is, the present invention is as follows.

[1] A method for producing a cloned non-human mammal, wherein a mammalian natural killer T cell is used as a donor cell.
[2] A method for producing a cloned non-human mammal, comprising introducing a nucleus of a mammalian natural killer T cell into a enucleated oocyte of a mammal.
[3] A clone comprising introducing a nucleus derived from a mammalian natural killer T cell into a enucleated oocyte of a mammal to form a reconstructed embryo, and transferring the reconstructed embryo to a host mammal A method for producing a non-human mammal.
[4] The method for producing a cloned non-human mammal according to any one of the above [1] to [3], wherein the natural killer T cell has been genetically manipulated to express a desired character. [5] The method for producing a cloned non-human mammal according to any one of [1] to [4] above, wherein the natural killer T cell and the enucleated oocyte are derived from the same kind of mammal.
[6] The method for producing a cloned non-human mammal according to any one of [1] to [4] above, wherein the natural killer T cell and the enucleated oocyte are derived from different mammals.
[7] The natural killer T cell is collected from a tissue selected from the group consisting of umbilical cord blood, peripheral blood, liver, bone marrow, spleen, and thymus, according to any one of [1] to [6] above. Of producing a cloned non-human mammal.
[8] A cloned non-human mammal produced by the method according to any one of [1] to [7] above.
[9] A fetus of a cloned non-human mammal obtained by the method according to any one of [1] to [7].
[10] A progeny of the cloned non-human mammal according to [8] above.

[11] The offspring of a cloned non-human mammal according to [10] above, wherein the TCRVβ chain expressed on T cells is substantially composed of a single TCRVβ chain repertoire. [12] The progeny of the cloned non-human mammal according to [11] above, wherein the single TCRV β chain repertoire is the same as the TCRV β chain repertoire of the NKT cell used as the donor cell.
[13] A progeny of a cloned non-human mammal according to [10] above, which has an allele containing Vα14-Jα281, which is a TCRα chain that has undergone gene rearrangement, and an increased number of NKT cells. .
[14] A cell, tissue, organ or product obtained from the cloned non-human mammal according to [8] above.
[15] A cell, tissue, organ or product obtained from the fetus of a cloned non-human mammal according to [9] above.
[16] A cell, tissue, organ or product obtained from the offspring of the cloned non-human mammal described in [10].
[17] The cell, tissue, organ or product according to any one of [14] to [16] above, which is immunologically identical to a donor cell.
[18] The cell, tissue, organ or product according to any one of [14] to [17], which is used for treatment, organ transplantation and / or cell transplantation.
[19] A method for producing a cloned mammalian embryo, characterized by using mammalian natural killer T cells as donor cells.
[20] A method for producing a cloned mammalian embryo, comprising introducing a nucleus of a mammalian natural killer T cell into a enucleated oocyte of a mammal.

[21] The method for producing a cloned mammalian embryo according to the above [19] or [20], wherein the natural killer T cell has been genetically manipulated to express a desired character.
[22] The method for producing a cloned mammalian embryo according to any one of [19] to [21] above, wherein the natural killer T cell and the enucleated oocyte are derived from the same kind of mammal.
[23] The method for producing a cloned mammalian embryo according to any one of the above [19] to [21], wherein the natural killer T cell and the enucleated oocyte are derived from different mammals.
[24] The natural killer T cell is collected from a tissue selected from the group consisting of umbilical cord blood, peripheral blood, liver, bone marrow, spleen, and thymus, according to any one of [19] to [23] above. To create a cloned mammalian embryo.
[25] A cloned mammalian embryo produced by the method according to any one of [19] to [24].
[26] The cloned mammalian embryo described in [25] above is cultured to the blastocyst stage or pre-blastocyst stage, and embryonic stem cells are separated from the resulting inner cell mass of the blastocyst stage or pre-blastocyst stage A method for producing a cloned mammalian embryonic stem cell.
[27] A cloned mammalian embryonic stem cell obtained by the method described in [26] above.
[28] A cloned mammalian differentiated cell, tissue, organ or product comprising culturing and inducing differentiation of the cloned mammalian embryonic stem cell according to [27] above under conditions capable of inducing desired cell differentiation How to make.
[29] A differentiated cell, tissue, organ or product of a cloned mammal obtained by the method described in [28] above.
[30] The differentiated cell, tissue, organ or product according to [29] above, which is immunologically identical to a donor cell.

[31] The differentiated cell, tissue, organ or product according to [29] or [30] above, which is used for treatment, organ transplantation and / or cell transplantation.

The cloned mammals, cloned mammal embryos obtained from the present invention, and cells, tissues, organs and products obtained from them have the same histocompatibility antigen (eg, HLA) as the donor, and have the same antigenicity. There is no immune rejection at the time of transplantation or administration. In addition, when NKT cells are used, it is not necessary to establish ES cells and tetracomplementation for cloning. This saves a lot of time and effort. Furthermore, when ES cells are established using the nuclei of NKT cells, the efficiency is about 6%, which is much higher than when conventional peripheral mouse T cells and B cells are used as donors. Moreover, it is very high compared with the efficiency (Non-patent Document 4) in which human ES cells can be established by autotransplantation. Furthermore, the rate of becoming an individual from the transplanted nucleus is about 1.5%, which is comparable to the success rate of cloning from ES cells (Non-patent Document 5), which has been considered to be obtained relatively easily so far.
By using the method of the present invention, it becomes possible to clone useful mammals (eg, transgenic sheep, cows, etc. that produce biopharmaceuticals in milk) easily because cloned mammals can be produced at a higher rate. Enables mass production and cost reduction of pharmaceuticals, contributing to the reduction of total medical costs.
Further, the progeny of the cloned mammal of the present invention is characterized in that the TCRVβ chain expressed on the T cell is substantially composed of a single TCRVβ chain repertoire, and more specifically, the NKT used as a donor cell. Identical to the cellular TCRV β chain repertoire. Furthermore, the number of NKT cells is relatively and absolutely increased compared to the wild type in the progeny having an allele containing the gene rearranged TCRα chain Vα14-Jα281. Based on these facts, the progeny of the cloned mammal of the present invention can be used as a model animal for pathological conditions such as autoimmune diseases, and also to analyze the significance of the role of NKT cells in immune control and the diversity of TCRVβ chain repertoire. Give.

Unless defined otherwise in the text, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned in this specification are herein incorporated by reference for the purpose of describing and disclosing constructs and methodologies described in, for example, publications that may be used in connection with the described invention. Incorporated into the book.
Hereinafter, the present invention will be described in detail. A summary of an example of the present invention is shown in FIG.
Natural killer T (NKT) cells used in the present invention are a type of lymphocyte that plays a regulatory role in the immune system, although its proportion is low. NKT cells have two antigen receptors, the T cell receptor (TCR) and the NK receptor. NKT cells express a specific repertoire different from normal T cells and NK cells. For example, with respect to the β chain of mice, more than 90% of NKT cells mainly express a limited repertoire of Vβ8, and other Vβ7 and Vβ2, and the α chain expresses a uniform Vα14-Jα281. This uniform TCRα chain is produced as a result of the Vα14 gene and Jα281 gene being selected from the V and J gene groups and rearranged on the genome during TCR gene rearrangement (Taniguchi et al., ( 2003) Annu Rev Immunol 21,483-513 The regulatory role of Valpha14 NKT cells in innate and aquired immune response). In humans, it is known to be a combination of non-polymorphic Vα24, which is highly homologous to mouse Vα14, and Vβ11 closely related to Vβ8.2. Such properties are suitable for tracking the origin of donor cells in the resulting cloned animals. The origin of NKT cells is not particularly limited, and umbilical cord blood, peripheral blood, liver, bone marrow of mammals such as primates including humans, rodents, rabbits, cats, dogs, horses, cows, sheep, goats and pigs, It can be collected from the spleen, lymph nodes, thymus, etc. As used herein, the term “primate” means any animal belonging to the group of mammals including, but not limited to, monkeys, apes and humans. Specifically, a single-cell suspension recovered from peripheral blood or liver homogenate is converted into α-galactosylceramide (α-GalCer or α) bound to a CD1d molecule that can be recognized by a TCR highly retained in NKT cells. -It can be selected and collected by FACS analysis using an antigen glycolipid such as GC). The NKT cells used in the present invention may or may not be activated. Cells that are not antigen-stimulated are preferred. Further, NKT cells obtained by culturing NKT progenitor cells in the presence of a factor conferring NKT cell differentiation-inducing ability may be used. NKT progenitor cells can be derived from fetal liver cells or peripheral blood or umbilical cord blood.

  The method for producing a cloned mammal of the present invention is characterized in that NKT cells are used as donor cells. Specifically, it is performed by nuclear transfer of NKT cells. The method of nuclear transfer is not particularly limited as long as it can be performed using the nucleus of the NKT cell as a donor cell nucleus. A cell nucleus may be introduced into an enucleated oocyte derived from a mammal of the same species as the cell nucleus, or a cell nucleus may be introduced into an enucleated oocyte derived from a mammal of a species different from the cell nucleus. The technique of nuclear transfer into enucleated oocytes derived from different species of mammals is useful for the resurrection of extinct species, the preservation and propagation of endangered species. Nuclear transfer to enucleated oocytes from the same species of mammal leads to pups more efficiently.

  As a method for introducing the nucleus of an NKT cell into an enucleated oocyte of a mammal, the method is not particularly limited as long as the nucleus of an unfertilized egg is finally replaced with the nucleus of an NKT cell that is a donor cell, In consideration of the size of the NKT cell, a method is preferably used in which the cell membrane of the NKT cell is scratched with a pipette or the like, and the damaged NKT cell is directly introduced into the enucleated oocyte using a micromanipulator or the like. Nuclear transfer may be performed by cell fusion between NKT cells and enucleated oocytes. More specifically, for example, according to the method reported by Wakayama et al. (Nature 394, 369-374 (1998)), Inoue et al. (Biol. Reprom. 69, 1394-1400 (2003)), and appropriately modified Can be implemented. When nuclear transfer is performed by fusing NKT cells and enucleated oocytes, they can be fused by an electrofusion method using a commercially available device.

  Mammalian oocytes subjected to enucleation can be obtained by superovulation treatment by hormone administration. Enucleation is carried out by using a micropipette in the oocyte to suck and tear off the nucleus and surrounding cytoplasm of the unfertilized oocyte, but the cytoskeleton is broken and the oocyte is destroyed as it is. Therefore, it is preferable to perform a treatment for eliminating the cytoskeleton of the unfertilized oocyte in advance. The disappearance of the cytoskeleton can be performed by using cytochalasin. Moreover, it is preferable to treat with cytochalasin or the like after nuclear transfer. An oocyte to be subjected to nuclear transfer may or may not be activated, but an activated state is preferable. The activation may be performed either before or after the nuclear transfer. The activation can be carried out by stimulation by an electrical method or a chemical treatment method, but is not particularly limited. It is particularly preferable to use naturally matured terminal oocytes. Activation can be performed, for example, by increasing the concentration of divalent cations in the oocyte and / or decreasing the degree of phosphorylation of cellular proteins in the oocyte. This is generally done by introducing a divalent cation, such as magnesium, strontium, barium, calcium, into the cytoplasm of the oocyte, for example in the form of an ionophore. Other methods of increasing the divalent cation concentration include the use of electric shock, ethanol treatment and cage chelator treatment. The degree of phosphorylation may be reduced by known methods, such as the addition of kinase inhibitors such as serine-threonine kinase inhibitors such as 6-dimethylaminopurine, 2-aminopurine and sphingosine. Alternatively, phosphorylation can be inhibited by introducing phosphatase into the oocyte.

  The nucleus of an NKT cell is introduced into an enucleated oocyte to form a reconstructed embryo, and the reconstructed embryo is transferred to the host mammal to generate the reconstructed embryo in the host mammal, thereby cloning Mammals can be obtained.

  As described above, when NKT cells are used as donor cells, cloned mammals can be obtained by somatic cell nuclear transfer without requiring the establishment of ES cells and sequential tetraploid embryo complementation. That is, NKT cells are predicted to have totipotency.

  As used herein, the term “totipotent” refers to a cell that gives rise to all cells in a developing cell mass, such as embryos, fetuses, and animals. In a preferred embodiment, the term “totipotent” means a cell that gives rise to any cell of an animal. Totipotent cells can give rise to any cell in the developing cell mass when used in a procedure that creates an embryo from one or more nuclear transfer stages.

  As used herein, the term “having totipotency” is distinguished from the term “having pluripotency”. The latter term refers to a cell that differentiates into a subpopulation of cells within the developing cell mass but cannot give rise to all cells in such a developing cell mass.

  As used herein, the term “embryo” or “embryonic” includes a developing cell mass that is not implanted in the endometrium of a host mammal. Thus, as used herein, the term “embryo” or “embryonic” refers to fertilized oocytes, cytoplasmic hybrids, cell masses during the preblastocyst stage, and / or to the endometrium of the host mammal. It may mean any other developing cell mass that is in the developmental phase prior to implantation. The reconstructed embryo means an embryo reconstructed with a donor cell-derived nucleus and an oocyte-derived cytoplasmic component obtained by nuclear transfer of a donor cell nucleus to an enucleated oocyte.

  Embryos may present multiple stages of cell development. For example, a single cell embryo can be called a zygote, a zygote is a solid, spherical cell mass that arises from a split embryo and can be called a morula, and an embryo with a split space is a blastocyst Can be called.

Transplantation of the reconstructed embryo into the host mammal can be performed according to a method commonly practiced in the art. Specifically, the reconstructed embryo is continuously cultured until reaching the 2-8 cell stage, the 2-8 cell stage embryo is transplanted into the host mammal, and the reconstructed embryo is further developed in the mammal to produce the cloned mammal. Create A medium suitable for culture and maturation of the reconstructed embryo is well known in the art, and is appropriately set according to the type of mammal from which the enucleated oocyte originates. Furthermore, the reconstructed embryo may be cultured together with feeder cells as desired, preferably on the feeder cell layer. The reconstructed embryo is cultured until it reaches a size suitable for transfer to the host mammal. Preferably, the cells can be cultured until they are about 2 to 400 cells, more preferably about 4 to 128 cells (in the case of mice, about 48 to 72 hours). This culture is performed under appropriate conditions, that is, in the presence of about 37 ° C. to 38.5 ° C. and about 5 to 7.5% CO ₂ , and the medium is appropriately changed. The host mammal is not particularly limited as long as it can generate a reconstructed embryo therein, but is preferably a mammal derived from the same species as the enucleated oocyte, more preferably a pseudopregnant female. Mammals. For example, in the case of mice, pseudopregnant female mice can be obtained by mating normal-period female mice with male mice castrated by vagina ligation or the like. A cloned mammal can be prepared by transferring the reconstructed embryo (cloned embryo) obtained by the above-mentioned method into the uterus, particularly in the fallopian tube, and then gesturing and giving birth to the created pseudopregnant mouse. . In order to ensure the implantation and pregnancy of cloned embryos, pseudopregnant mice that become foster parents are selected from a group of female mice in the same sexual cycle as the female mice from which the oocytes to be enucleated are collected. It is preferable to do.

  As used herein, the term “fetus” means a developing cell mass that is implanted in the endometrium of a host mammal. The fetus may contain distinct features such as genital ridges. Genital ridge is a feature that is easily identified by those skilled in the art and is a recognizable feature in the fetus of many animal species. Fetal cells may mean any cells isolated from and / or generated from a fetus, or cells derived from a fetus. The fetus of the cloned mammal of the present invention can be obtained by collecting the reconstructed embryo into a temporary parent and then collecting it at an arbitrary stage in the above-described process of creating the cloned mammal.

  The offspring of the cloned mammal of the present invention can be obtained by crossing a cloned mammal developed from the reconstructed embryo with a second mammal. The second mammal may be a normal mammal (not a clone) of the same species as a cloned mammal (also referred to as the first mammal for convenience), or a cloned mammal generated from a cloned embryo or its progeny. . It may be a transgenic mammal described later or a cloned mammal generated from a transgenic mammal embryo or a progeny thereof. In the present invention, the term “offspring” refers to the present invention of the offspring generation (F1) whose parent is the cloned mammal of the present invention, the offspring generation (F2) whose parent is F1, and the offspring generation (F3) whose parent is F2. Any generation can be used as long as it originates from a cloned mammal. In the present invention, cells, tissues or organs can be arbitrarily obtained using the above cloned mammals, fetuses of cloned mammals, and descendants of cloned mammals. It is not necessary to collect and collect cells, tissues or organs by a particularly limited method, and is usually performed by a method practiced in the art. Furthermore, the products secreted, produced and extracted from the obtained cells, tissues or organs are also within the scope of the present invention. The products obtained from cloned mammals are as diverse as those obtained from target mammals, but include glycoproteins, neuropeptides, immunoglobulins, enzymes, peptides and hormones. More specifically, human α1 antitrypsin, human blood coagulation factor VIII, tPA (tissue specific plasma activator), antithrombin III, protein C, fibrinogen, human blood coagulation factor IX and the like can be mentioned.

  Cells, tissues, and organs derived from cloned mammals, fetuses, and offspring obtained as described above have the same histocompatibility antigen (eg, HLA in humans) as NKT cells that are donor cells. When cells, tissues, and organs derived from cloned mammals, their fetuses and offspring are transplanted into mammals that are donor cells, they become immune-tolerated without being recognized as non-self, and immune rejection occurs. Does not happen.

Further, the progeny of the cloned mammal of the present invention have the following characteristics (details will be described in Examples below).
(1) The TCRVβ chain expressed in an individual is substantially the same as the single TCRVβ chain repertoire, particularly the TCRVβ chain repertoire of NKT cells used as donor cells.
(2) The number of NKT cells is increasing.
The rearrangement of TCR genes plays an important role in the repertoire formation of a huge number of lymphocytes involved in the differentiation and selection of T cells. For example, expression of a recombination activating gene (RAG) is essential for the rearrangement of these genes, and it has been clarified that severe combined immunodeficiency and Omen's syndrome develop due to abnormalities and defects in RAG.
As described in (1) above, there is no report other than the TCR transgenic animal for the phenomenon that the TCR within an individual is limited to one specific type, and use the offspring of a cloned mammal having such characteristics. Thus, differentiation and selection of only one kind of T cell clone, which is originally one in 100,000 or one million, can be observed at the individual level. For example, T cells expressing a single TCRVβ chain express αβ type TCR and suppress the expression of γδ type TCR. Such a phenomenon is similar to a phenomenon observed in intestinal diseases such as colitis, and therefore, the offspring of the cloned mammal of the present invention is also useful as a model animal such as colitis. Furthermore, the number of NKT cells is increased in the progeny of cloned mammals having an allele containing Vα14-Jα281, which is a TCRα chain having undergone gene reconstitution according to the present invention. An increase in the number of NKT cells is observed both in the ratio to the total spleen cells (the ratio of NKT cells) and in the absolute number. Since NKT cells are cells having a function of controlling the immune system, the progeny of the cloned mammal of the present invention having a larger number of NKT cells can be used to study diseases and / or pathologies in which NKT cells are involved, particularly It is expected to greatly contribute to the elucidation of the pathogenesis of autoimmune diseases and allergic diseases, transplanted bone marrow rejection, and tumor immunity. More specific diseases and / or conditions include inflammatory conditions in humans and animals, various pains, collagen diseases, autoimmune diseases, various immune diseases, particularly inflammation and pain in joints and muscles (rheumatoid arthritis, rheumatoid arthritis) Spondylitis, osteoarthritis, uric acid arthritis, etc.), inflammatory condition of the skin (eg, eczema), inflammatory condition of the eye (eg, conjunctivitis), pulmonary disorders with inflammation (eg, asthma, bronchitis), digestion with inflammation Genital conditions (aphthous ulcer, Crohn's disease, atrophic gastritis, wart gastritis, ulcerative colitis, steatosis, localized ileitis, irritable bowel syndrome, etc.), gingivitis, inflammation after surgery or injury , Pain, swelling), fever associated with inflammation, pain, other conditions, transplant rejection, systemic lupus erythematosus, scleroderma, polymyositis, polychondritis, nodular periarteritis, ankylosing spondylitis, Inflammatory chronic kidney condition ( Globe nephritis, lupus nephritis, membranous nephritis, etc.), rheumatic fever, Sjogren's syndrome, Behcet's disease, thyroiditis, type I diabetes, dermatomyositis, chronic active hepatitis, myasthenia gravis, Graves' disease, multiple sclerosis, primary Biliary cirrhosis, autoimmune blood diseases (hemolytic anemia, true erythrocytic anemia, idiopathic thrombocytopenia, aplastic anemia, etc.), uveitis, contact dermatitis, psoriasis, Kawasaki disease, type I allergy Diseases involving reactions (allergic asthma, atopic dermatitis, urticaria, allergic conjunctivitis, hay fever, etc.), shock (septic shock, anaphylactic shock, adult respiratory distress syndrome, etc.), sarcoidosis, Wegener's granuloma Disease, Hodgkin's disease, cancer (lung cancer, stomach cancer, colon cancer, stomach cancer, liver cancer, etc.), viral disease (hepatitis) and the like.

  The present invention also provides a method for producing a cloned mammalian ES cell using the above-described reconstructed embryo (also referred to as a cloned embryo or a cloned mammalian embryo) and a cloned mammalian ES cell obtained thereby. ES cells include pluripotent cells, preferably isolated from embryos maintained in in vitro cell culture. ES cells can be cultured with or without feeder cells, but feeder cells are preferably used. As the feeder cells, those commonly used in the art can be used. For example, in the case of mouse ES cells, mouse embryo-derived fibroblasts can be used. Clonal mammalian ES cells can be established from embryonic cells isolated from embryos at any stage of development, including embryos at the blastocyst stage and embryos at the preblastocyst stage. More specifically, the nucleus of an NKT cell that is a donor cell is introduced into an enucleated oocyte, and the resulting cloned mammalian embryo is cultured to the blastocyst stage and / or the preblastocyst stage, and the resulting internal It is made by isolating from a cell mass. As used herein, the term “inner cell mass” means a cell that gives rise to the embryo body. The cells that line up outside the blastocyst are called embryo trophoblasts. Methods for isolating inner cell mass from embryos are known to those skilled in the art.

  The cloned mammalian ES cells can be cultured and induced under conditions capable of inducing desired cell differentiation, and desired differentiated cells, tissues and organs can be produced. Conditions that can induce desired cell differentiation should be set according to the type and degree of differentiation, and can be easily set by those skilled in the art. For example, embryonic stem cells are induced to differentiate into hematopoietic stem cells, and further differentiated to finally produce blood cells such as red blood cells and white blood cells. Alternatively, it is possible to differentiate into neural stem cells and induce differentiation into individual neural cells.

  Confirmation that the fetus and offspring obtained in the present invention are derived from NKT cells, which are donor cells, is performed by examining the obtained fetus and offspring genes, specifically the TCR gene. NKT cells are reconstituted with the TCR gene, and as a result, for example, in mice, Vα14-Jα281 is characteristically expressed in the α chain as described above. Furthermore, it has a Vβ chain that has undergone gene rearrangement. The genomic DNA of the cloned mammalian fetus and offspring inheriting the genetic information of the NKT cell is subjected to Southern blot analysis using the TCRVα14 probe and / or the TCRVβ probe, preferably using both the TCRVα14 probe and the TCRVβ probe, The cloned mammal fetus and offspring obtained by confirming whether the TCR gene has been reconstituted, in particular, whether or not it exhibits the same TCR reconstitution pattern as the NKT cells used as donor cells are donors It can be determined whether it is derived from a somatic NKT cell. In addition, whether or not the reconstructed TCR gene is inherited can also be confirmed by the PCR method. A specific confirmation procedure will be described later in the embodiment.

  The above-described method for producing a cloned mammal of the present invention can also be used for cloning a genetically engineered mammal or a transgenic mammal. Specifically, a mammalian NKT cell that has been genetically manipulated to express a desired trait is used as a donor cell, and more specifically, genetic manipulation is performed to express a desired trait. This is carried out by introducing the nucleus of a mammalian NKT cell into an enucleated oocyte to form a reconstructed embryo (transgenic embryo) and transferring the reconstructed embryo into a host mammal. “Transgenic embryo” means an embryo containing a heterologous nucleic acid into which one or more cells have been introduced by human intervention. A transgene can be introduced directly or indirectly into a cell by introduction into a cell precursor, by deliberate genetic manipulation, or by infection with a recombinant virus. In the transgenic embryos described herein, the transgene expresses a structural gene that allows the cell to exhibit the desired trait. However, transgenic embryos in which the transgene is silent are also included. The transgenic embryo is the same as that obtained in the process of producing the cloned mammal described above, except that the nucleus of the donor cell is the nucleus of a cell that has been genetically manipulated to express a desired trait. It is created in the same way.

  The transgenic mammal of the present invention also includes a germline transgenic animal to which the ability to transmit genetic information to offspring is imparted by introducing genetic changes or genetic information into the germline. The

  The term “gene” refers to a DNA sequence that includes regulatory and coding sequences necessary for the production of a polypeptide or precursor. A polypeptide can be encoded by a full-length coding sequence or can be encoded by any portion of the coding sequence so long as the desired activity is retained.

  The term “transgene” broadly refers to any nucleic acid that is introduced into the genome of an animal, probably a gene or DNA having a sequence that is not normally present on the genome, but is normally transcribed and present in a given genome. This includes, but is not limited to, genes that are not translated (expressed) or any other gene or DNA that is desired to be introduced into the genome. Transgenes may include genes that are normally present on non-transgenic genomes but whose expression is desired to be altered, or genes that are desired to be introduced as altered or variant. A transgene may be specifically directed to a limited locus, may be randomly integrated into the chromosome, or may be DNA that replicates extrachromosomally. The transgene may include one or more transcriptional regulatory sequences and any other nucleic acid, such as an intron, that may be necessary for the proper expression of the selected nucleic acid. The transgene may be a coding sequence or a non-coding sequence, or a combination thereof. A transgene may contain regulatory elements that have the ability to drive expression of one or more transgenes under appropriate conditions.

  The term “structural gene capable of exhibiting a desired trait” means a structural gene that expresses, for example, a protein or antisense RNA that exhibits the desired trait (eg, exhibits biological activity). The structural gene is any source known in the art, including plant, fungal, animal, bacterial genome or episome, eukaryotic nuclear DNA or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA May be derived in whole or in part. Structural gene sequences include, for example, receptors, enzymes, cytokines, hormones, growth factors, immunoglobulins, cell cycle proteins, intracellular signaling proteins, membrane proteins, cytoskeletal proteins, or reporter proteins (eg, green fluorescent protein (GFP), In some cases, it encodes a polypeptide such as β-galactosidase or luciferase. Furthermore, the structural gene may be a gene related to a specific disease or disorder such as cardiovascular disease, neurological disease, reproductive disorder, cancer, eye disease, endocrine disorder, pulmonary disease, metabolic disorder, autoimmune abnormality, aging and the like.

  A structural gene may contain one or more modifications in the coding or untranslated region that can affect the biological activity or chemical structure, expression rate, or expression control mode of the expressed product. Such modifications include, but are not limited to, one or more nucleotide mutations, insertions, deletions, and substitutions. A structural gene may constitute a contiguous coding sequence, or may contain one or more introns linked to an appropriate splice junction. The structural gene may encode a fusion protein.

  Transgenic mammalian ES cells can also be produced using transgenic embryo cells by a method according to the above-described method for producing cloned mammalian ES cells of the present invention. The obtained transgenic mammalian ES cells can be cultured and induced under conditions capable of inducing desired cell differentiation, similar to the above-described cloned mammalian ES cells, and the desired differentiated cells, tissues and organs can be induced. Can be created. The obtained differentiated cells, tissues and organs have a structural gene capable of exhibiting a desired trait, and by expressing the gene, differentiated cells, tissues and organs having the desired trait, or products produced therefrom are used. Obtainable. Recovery of cells, tissues and organs, or products recovered from them can be performed using a method commonly practiced in the art, and is appropriately set according to the desired character. In addition, the methods described in the present invention can be used to generate transgenic primate ES cells capable of expressing genes associated with specific diseases. Therefore, many human diseases can be treated by the method described in the present invention and the resulting ES cells.

  As described above, the present invention provides not only a method for producing a cloned mammal, but also the possibility of producing a transgenic cloned mammal. Such transgenic mammals can also be used as research models for serious human diseases and as models for evaluating the efficacy of gene and cell therapeutic strategies. Furthermore, the stem cells obtained by the present invention can be said to be extremely important for the study of many diseases (eg, aging, AIDS, cancer, Alzheimer's disease, autoimmune disorders, metabolic disorders, obesity, organogenesis, psychiatric disorders, and reproduction). .

  The cloned mammals and transgenic animals of the present invention are considered to have a single TCRVβ chain and a relative and absolute increase in the number of NKT cells.

  Using cloned animals and transgenic animals, it is possible to discover the onset mechanism of diseases and to create and optimize molecular medical treatments. For example, mammals created by gene knockout for specific genes can facilitate the discovery of treatments for arteriosclerosis, inborn errors of metabolism, and other fetal and neonatal diseases that cause cancer, heart disease and stroke. Such animals also have diabetes, liver damage, kidney damage, artificial organ development, wound healing, heart attack damage, brain damage due to stroke, spinal cord injury, memory loss, Alzheimer's disease, and other dementia, muscle and Suitable for evaluating and improving cell therapy of diseases, including nerve damage.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail according to an Example, these Examples do not limit the scope of the present invention at all. All publications cited throughout this application are incorporated herein by reference. In addition, the reagents, devices, and materials used in the present invention are commercially available unless otherwise specified.

Example 1 Preparation of NKT Cell-Derived Cloned Mice Mononuclear cells were obtained from the liver of 8-week-old to 23-week-old (C57BL / 6 × 129 / Sv-ter) F1 female mice by Percoll (trade name, Amersham) specific gravity centrifugation. I got it. CD1d tetramer (α-GC loaded CD1d-tetramer to which these cells were prepared in-house, and phycoerythrin (PE) -α-galactosylceramide (α-GC); Matsuda, JL, OV Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, CR Wang, Y. Koezuka, and M. Kronenberg. 2000. Tracking the response of natural killer T cells to a glycolipidantigen using CD1d tetramers. J Exp Med 192: 741-754.) And fluoroscein isothio
The cells stained with cyanate (FITC) -anti-TCRVβ antibody (H57) (PharMingen) were used as NKT cells (that is, NKT cells are defined as α-GC loaded CD1d-tetramer ⁺ / TCRβ ⁺ )
These cells were purified as a PE ⁺ / FITC ⁺ cell population using a fluorescent activated flow cytometer (FACS) (Cytomation MoFlo (registered name)) and used as a donor for nuclear transfer. The NKT cell sorting was repeated twice, and NKT cells were obtained with a purity of 99% or more.

Recipient eggs were removed from ovulation inducing female mice treated with ovulation by ovarian flushing, cumulus cells were removed by hyaluronidase treatment, and then enucleated using an piezo micromanipulator under an inverted microscope and used for nuclear transfer . While aspirating NKT cells into a pipette, the cell membrane was damaged by physical stimulation of a piezo micromanipulator. Subsequently, a nuclear transfer was performed by stenosis in the zona pellucida and the cell membrane of the enucleated ovum using a piezo micromanipulator and injecting the NKT cell nucleus into the cytoplasm. Similarly, using T cells, reconstructed eggs obtained by injecting T cell nuclei into the cytoplasm were obtained and used as controls.
One hour after nuclear transfer, the reconstructed ovum was activated for 1 hour in KSOM medium containing cytochalasin and strontium, then cultured for 5 hours in KSOM medium containing only cytochalasin, and further cultured in KSOM medium and observed over time. . After 24 hours of culture, most of the NKT cell-derived and T-cell-derived reconstructed embryos were in the 2-cell stage state, which seems to be in the G0 stage. When cultured further for 24 hours (total 48 hours), the reconstructed embryo obtained by transplanting the T cell-derived nucleus stopped at the 2-cell stage, but was obtained by transplanting the NKT cell-derived nucleus. The reconstructed embryo had transitioned to the 4-cell stage. When the culture was further continued (72 hours in total), 71% of the reconstructed embryos transplanted with nuclei derived from NKT cells became Morula blast cysts, whereas that of reconstructed embryos transplanted with nuclei derived from T cells The proportion was only 12%.
The cells were cultured for 48 to 72 hours (each corresponding to a 4-cell stage, Morula blast cyst) and returned to the oviduct of ICR mice on the first day of pseudopregnancy. After 19 days, pups were removed by cesarean section (FIG. 1).
When 272 embryos were transplanted into pseudopregnant mice, 4 mice were born (approximately 1.5% probability). There were also 13 conceptus products (approximately 4.8% probability). These results are very similar to those using ES cells as donors, and differentiated somatic cells, NKT cells, may be as effective as ES cells in reprogramming the genome for mouse reproduction. all right. All cloned placentas showed moderate or high placental hyperplasia observed in mouse somatic cell cloning. The above results are summarized in Table 1 and FIG.

In order to prove that the obtained cloned mice were derived from NKT cells, Southern blotting was performed to detect Vα14, a subunit of TCR unique to the cells.
NKT cells have a combination in which the TCRVα chain is only Vα14-Jα281. If the created cloned mouse is derived from NKT cells, the genomic DNA of this mouse, as well as the placenta, should have a genomically reconstructed genomic DNA of Vα14-Jα281. Similarly, the Vβ chain should have genomic DNA that has undergone gene rearrangement. The Southern blot probe was prepared as follows.
Preparation of probe for Southern blot PCR reaction was performed using genomic DNA extracted from the tail of C57BL / 6 mice as a template using the following primer set. Each primer was prepared using custom primer synthesis from In Vitrogen.
The base sequence of the PCR product has been confirmed by DNA sequencing. <PCR primer for TCRVα14 Southern probe preparation>
Primer sequence 1: 5′-CGCTTGTGCACATTTGTTTCT-3 ′ (SEQ ID NO: 1)
Primer sequence 2: 5′-TAAGTTTCTGGGGAGCATGG-3 ′ (SEQ ID NO: 2)
<PCR primer for TCRVβ Southern probe preparation>
Primer sequence 3: 5′-GGGGCTGTGAACCAAGACAC-3 ′ (SEQ ID NO: 3)
Primer sequence 4: 5′-TACCTTTCGCTCCCTTTCAAAAGACC-3 ′ (SEQ ID NO: 4)
FIG. 2 shows the position of the TCRVα14 Southern probe on the genome, and FIG. 3 shows the position of the TCRVβ Southern probe on the genome. FIG. 2 schematically shows various Vα fragments near the Vα14 gene. The exon part is drawn as a square. FIG. 3 schematically shows the Vβ, D, J, and C segments.
After digesting 5-30 μg of genomic DNA with EcoRI (in the case of TCRVα14) or BamHI (in the case of TCRVβ), the DNA fragments were separated by electrophoresis and transferred onto nylon membrane Hybond N + (trade name, Amersham). After UV cross-linking, prehybridization was performed with PerfectHyb (registered name, TOYOBO) solution (68 ° C., 60 minutes), and RI-labeled TCRVα14 Southern probe or TCRVβ Southern probe was added by Reprime II (Amersham). Hybridization was performed at 68 ° C. for 16 hours. Thereafter, the plate was washed twice with 2 × SSC, 0.1% SDS (68 ° C.) solution for 5 minutes and further twice with 0.1 × SSC, 0.1% SDS (68 ° C.) solution for 15 minutes. The washed nylon membrane was exposed with Image plate (Fuji Film) and analyzed with the company's Image plate Reader BAS 2500.

In all the born mice, rearrangement of the Vα14 chain was observed. The results are shown in FIG.
Since genomic DNA having germline configuration in which gene rearrangement has not occurred has no rearranged product of Vα-Jα, Southern blotting using the Vα14 probe has an allele derived from 129 / Sv-ter mice. In this case, a 2.5 kb band is generated (FIG. 4, lane 2), and an allele derived from C57BL / 6 mice results in an approximately 13 kb band (FIG. 4, lane 1). This is because the fragment produced when digested with EcoRI differs depending on whether it is derived from 129 / Sv-ter or C57BL / 6. C57BL / 6x129 / Sv-ter mice from which donor cells are derived have both alleles, producing both an approximately 2.5 kb band and an approximately 13 kb band (Figure 4, lane 3). In cloned mice # 1, # 3, and # 4, 8 kb bands are detected by recombination in genomic DNA having the Vα14 locus after gene rearrangement, but the bands of about 13 kb and 2.5 kb remain as they are. (FIG. 4, lane 4 and lane 5, lane 8, lane 9). When considered together with the results of the nucleotide sequence determination of the TCRVα chain described later, it can be seen that this 8 kb band is derived from the C57BL / 6-derived allele in the cloned mouse. In addition, it is considered that a band of about 13 kb remains after gene rearrangement because two Vα14 genes exist in the C57BL / 6 allele, and one of them is a pseudogene.
In clone mouse # 2, the 8 kb band was detected by recombination in the genomic DNA having the Vα14 locus after gene rearrangement, and the approximately 13 kb band remained as it was, but the 2.5 kb band remained unchanged. The band disappeared. This suggests that gene rearrangement also occurred in the allele derived from 129 / Sv-ter (FIG. 4, lane 6 and lane 7). From the result of the DNA base sequence (FIG. 6), it was revealed that 129 / Sv-ter-derived Vα14-Jα281 was used in cloned mouse # 2.

  Similarly, TCRVβ Southern blotting showed that one or both alleles were rearranged. In the genomic DNA of germline configuration in which gene rearrangement has not occurred, only a 10.4 kb band is generated, whereas those having loci that have completed gene rearrangement generate bands of various sizes by recombination. The results are shown in FIG.

  In the process of creating a cloned mouse, only an empty placenta may be obtained without reaching a litter. The resulting genomic DNA of the empty placenta was extracted and similarly subjected to Southern blot analysis of TCRVα14 and TCRVβ. As a result, the Southern blot pattern of genomic DNA derived from the empty placenta was consistent with the Southern blot pattern of genomic DNA derived from the tail of the cloned mouse, as well as the Vα14 chain and Vβ chain. (However, a band derived from a temporary mother may be observed in genomic DNA derived from placenta. In the case of TCRVβ, it is 10.4 kb).

Furthermore, PCR was performed using these genomic DNAs as templates, and the DNA sequences of the Vα and Jα chains were examined. Genomic DNA was extracted from the tail of cloned mice (clone mice # 1, # 2 and # 4) or its placenta, and the sequence of Vα14-Jα281 was amplified by PCR. If all these genomic DNA alleles are configured prior to gene rearrangement (germline configuration), no PCR product will be produced. This is because the Vα14 and Jα281 gene fragments are separated by several megab or more and cannot be amplified by PCR. However, when the gene is rearranged, a PCR product of about 330 bp should be generated (see FIG. 5). Furthermore, since sequence polymorphism exists in the Vα14 gene, if the DNA sequence of the obtained product is determined, it can be distinguished whether the Vα is derived from 129 / Sv-ter or C57BL / 6 (donor NKT cells are C57BL / 6). And 129 / Sv-ter) (see FIG. 6).
PCR primers for detecting Vα14-Jα281 are as follows. FIG. 5 schematically shows the position of each primer on the genome.
<PCR primer for detection of Vα14-Jα281>
Primer sequence 5: 5′-CCCAAGTGGAGCAGAGTCCT-3 ′ (SEQ ID NO: 5)
Primer sequence 6: 5′-AGGTATGACAATCAGCTGGAGTCC-3 ′ (SEQ ID NO: 6)
PCR was performed using a combination of the genomic DNA from the placenta of the cloned mouse and the tail as a template (primers 5 and 6) (for 50 ng of template DNA, using ABI's AmpliTaq Gold at a primer concentration of 0.2 μM, The sample was treated at 94 ° C. for 10 minutes, and a cycle of 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 1 minute was performed 36 times). As a result, a PCR product of about 330 bp was obtained (FIG. 5, PCR diagram, lane) 2 and lane 9). On the other hand, no product was obtained as expected with the genomic DNA of the control C57BL / 6, 129 / Svj, or blood stem cell-derived cloned mouse (FIG. 5). When the DNA base sequence of this PCR product was determined, it was found that it had in-frame TCRVα14-Jα281 with the gene rearranged as shown in FIG. Clone mice # 1 and # 4 were C57BL / 6 type, and clone mouse # 2 was 129 / Sv-ter type.

  Unlike the TCRVα chain, the TCRVβ chain is not uniquely determined in NKT cells. In order to investigate which Vβ chain is used, PCR was performed using primers capable of detecting various Vβ chains and the tail of cloned mouse # 1 and placental genomic DNA as a template to determine the DNA base sequence ( (See Figure 3 for the position of each primer on the genome). The combination of PCR primers used is an arbitrary one from the group A of primers shown below and an arbitrary one from the group B of primers. In this case, as in the case of the TCRVα chain, a PCR product is obtained from an allele having a TCRVβ locus where gene rearrangement has been completed, whereas a product is not obtained from a normal allele having a germline configuration.

<PCR primer for TCRVβ detection>
Primer group A (TCRVβ side);
Primer sequence 7 (for Vβ2): 5′-CACGGGTCACTGATACGAGAGC-3 ′ (SEQ ID NO: 7)
Primer sequence 8 (for Vβ3): 5′-TGAGTGTCCTTCAAACTCACC-3 ′ (SEQ ID NO: 8)
Primer sequence 9 (for Vβ4): 5′-AAACCATTTAGACCTTCAGAT-3 ′ (SEQ ID NO: 9)
Primer sequence 10 (for Vβ5): 5′-AGTTTGATGAACTATCACTCTG-3 ′ (SEQ ID NO: 10)
Primer sequence 11 (for Vβ6): 5′-GGGCAAAAACTGACCTTGAA-3 ′ (SEQ ID NO: 11)
Primer sequence 12 (for Vβ7): 5′-TCTCACCGGAAGAAGCGGGAGC-3 ′ (SEQ ID NO: 12)
Primer sequence 13 (for Vβ8): 5′-GATACAAGGCCTCCAGACCA-3 ′ (SEQ ID NO: 13)
Primer sequence 14 (for Vβ11): 5′-GCCCAATCAGTCGCACTCAAC-3 ′ (SEQ ID NO: 14)
Primer sequence 15 (for Vβ12): 5′-CATCCTCTCTCACTCTGAAGA-3 ′ (SEQ ID NO: 15)
Primer group B;
Primer sequence 16: 5′-GAAGGGACGAACTCTGTCTTACCTT-3 ′ (SEQ ID NO: 16)
Primer sequence 17: 5′-TGAGAGCTGTCTCCTACTACTCGATT-3 ′ (SEQ ID NO: 17)

  The results of base sequence determination are shown in FIG. Cloned mouse # 1 had the framework of Vβ8S2-D1-Jβ2S5 in gene reconstructed, and cloned mouse # 2 was Vβ8S3-D1-Jβ1S4. Furthermore, when the peripheral blood of cloned mice # 1 and # 2 was analyzed by FACS for Vβ phenotype, almost all of them were TCRVβ8 positive unlike that of donor cells (FIG. 12). This indicates that allelic exclusion has occurred in both cloned mice. These cloned mice have no apparent abnormality and are still normal except for a slight obesity tendency even after 12 months from birth (as of June 2005).

  From the above results, it was confirmed that the origin of the cloned mouse obtained in the present invention was peripheral NKT cells after gene rearrangement.

Example 2: Establishment of ES cell line from NKT cells ES cell establishment was attempted by the direct nuclear transfer method described in Example 1 using NKT cell-derived nuclei. 97 NKT cells were injured with a pipette and transplanted into an enucleated egg as described above. Prepare cells in advance by washing with ES medium (DMEM containing FCS, L-glu, NEAA, P / S, LIF and 2ME) 60 hours after transplantation (Day 2.5, 8-cell state, embryo) The seeded embryonic fibroblasts were reseeded (1 embryo / culture dish). Thereafter, the cells were cultured until the inner cell mass (ICM) was sufficiently grown under conditions of 37 ° C. and 7% CO ₂ (about 7 to 10 days), and cells having 16 ICMs were obtained. . Further, the ICM was divided into small pieces using a syringe and a needle, and the culture was continued until ES colonies were confirmed. Eleven ES cell lines were obtained by this series of steps. Of these, 5 were differentiated, but 6 ES cell lines could be established. Furthermore, when the ability of these 6 ES cell lines to form chimeric mice was examined, the ability to produce chimeric mice for 4 ES cells was confirmed. A summary of the course is shown in Table 2.

  Further, the TCRVα gene locus of the obtained ES cells was analyzed by PCR in the same manner as in Example 1 (FIG. 5, lanes 3 to 8). As a result, a 330 bp PCR product was obtained, and it was proved that gene rearrangement was performed. Further, Southern blot analysis was performed in the same manner as in Example 1. As a result, it was confirmed that the TCRVα14 chain was reconstituted with the same gene as that of the donor cell NKT cell, and that the TCRVβ chain was also reconstituted (FIG. 8, lanes 1 to 6).

Example 3: Offspring of cloned mice (reproductive ability of cloned mice)
In order to examine the reproductive ability of NKT cell-derived cloned mice, cloned mouse # 1 was mated with female mice of ICR, C57BL / 6 strain. As a result, 47 children were born in three months. From this fact, it is judged that the reproductive ability of this cloned mouse is normal. Southern blot analysis was performed on the genomic DNA in these children (offspring F1). F1 which inherited the 8 kb band derived from the TCRVα14 derived from the cloned mouse-derived gene rearranged was found in male No. 1 and female No. 6, and the allele TCRVα14 chain of the cloned mouse after gene rearrangement was inherited in germ cells. (Fig. 9, upper row, arrow). The alleles containing TCRVβ derived from the parent clone mouse # 1 are separated into about 9 kb and 8 kb, respectively (see FIG. 4), but each F1 is a 10.4 kb band derived from the mother mouse (FIG. 9, lower panel). , White arrowhead) and a band of about 9 kb or 8 kb (FIG. 9, bottom row, black arrowhead). For example, male No. 1 and female Nos. 3, 6 and 7 inherit a band of about 9 kb, males 2-6, females No. 1, 2, 4, 5 and 8 inherit an band of about 8 kb. . That is, it was found that one allele including the genome rearranged genome of the father clone mouse was inherited one by one for the TCRVβ chain (FIG. 9). These facts proved that these genetically rearranged genomic DNAs are transmitted to offspring.

The cell surface antigens of TKT and NKT cell progeny of NKT cloned mice having the rearranged alleles were analyzed by FACS. Analysis was performed on the spleen and liver of NKT cloned mouse F1. The spleen was crushed on two glass slides to form single cells, and red blood cells were removed and used for staining. The liver was ground with a metallic mesh having a mesh diameter of 75 μm, prepared to a mononuclear cell by a density gradient of Percoll, and then erythrocytes were removed and used as a single cell for staining. These cells were cloned by staining with an anti-TCRVβ antibody (the same as that used in Example 1) that recognizes all TCRVβ and an antibody that recognizes only TCRVβ8 (PharMingen product number 553861) and performing FACS analysis. The proportion of TCRVβ8 in mouse offspring was examined. The results are shown in the upper part of FIG. Similarly, the spleen and liver are decomposed to single cells, then stained with CD1d tetramer (same as used in Example 1) and anti-TCRVβ antibody (same as used in Example 1), and FACS analysis The proportion of NKT cells in cloned mouse offspring was examined. The results are shown in the lower part of FIG. The ratio of expressing TCRVβ8 was almost 100% in the mouse (FIG. 10, 2 panel) that inherited TCRVβ8 in frame, whereas the wild type was more than 20% of T cells in the spleen. A similar trend was seen in the liver. In wild-type spleen cells, the proportion of NKT cells defined by α-GC loaded CD1d-tetramer ⁺ / TCRβ ⁺ was approximately 0.5% of total spleen cells, but inherited gene-reconstituted Vα14-Jα281. It was up to about 13% for the ones (panels in Figs. 10 and 3). In this mouse, the total number of spleen cells was about half that of the wild type, but the absolute number of NKT cells was about 13 times (FIG. 10).
Based on the above results, the descendants of cloned mice that inherited the in-frame TCRVβ chain that had undergone gene reconstitution were found that the TCRVβ chain expressed on the T cell was almost entirely defined by the in-frame unique Vβ chain. It consists of a repertoire. That is, so-called allelic exclusion as observed in transgenic mice is observed. Furthermore, the progeny that inherited TCRVα14-Jα281 has an absolute number and ratio of NKT cells that are 10 to 20 times higher than those of the wild type. These results are the results of F1 with ICR, but the same tendency was observed with F1 crossed with C57BL / 6.

Example 4: Offspring of cloned mice (function of NKT cells)
In this example, the function of NKT cells of descendants of cloned mice was confirmed. Cytokine production when stimulated with α-galactosylceramide (α-GC) was examined in vitro and in vivo.
F1 was prepared by crossing cloned mouse # 1 with female C57BL / 6. Thereafter, this F1 male, which contained Va14-Ja281 that had been reconstructed, was SPFed by IVF (in vitro fertilization) and again crossed with female C57BL / 6 to obtain offspring (Vα14-Jα281 mice). Experiments were performed using 3-month-old Vα14-Jα281 mice and age-matched litters (control mice).
Cytokine production by in vivo stimulation (method)
In vivo stimulation was performed by intraperitoneally administering α-GC to mice at a dose of 2 μg / mouse. Serum of mice stimulated in vivo was collected at a predetermined time, and the concentrations of various cytokines in the serum were measured with a Bio-Plex Suspension Array System (BioRad, Hercules, CA).
(result)
The results are shown in FIG. Cytokines (IL (interleukin) -2,4,10, GM-CSF (granulocyte / macrophage colony-stimulating factor), IL-) for Vα14-Jα281 mice (mice that inherit genetically reconstructed Vα14-Jα281 heterogeneously with germline) Enhancement of 1β, TNFα (tumor necrosis factor α), IFN (interferon) -γ) production ability was confirmed. In addition to the increase in the absolute number and ratio of NKT cells confirmed in Example 3, it was confirmed that the function was also maintained.

IL-4 and IFN-γ production by in vitro stimulation (method)
Dendritic cells (DC) were obtained from the spleen of mice (Vα14-Jα281 mice or littermate control mice) using CD11c microbeads (Miltenyi). Dendritic cells derived from each mouse were stimulated with α-galactosylceramide (200 ng / mL) for 6 hours and mixed with spleen cells (SPC) derived from each mouse. These cells were co-cultured for 72 hours, and production of IFN-γ and IL-4 was measured using an ELISA kit (Genzyme TECHNE, Menapolis, MN).
(result)
The results are shown in FIG. It was confirmed that IL-4 and IFN-γ producing ability was increased in spleen cells derived from Vα14-Jα281 mice compared to control mice.

SEQ ID NO: 1: PCR primer for preparing TCRVα14 Southern probe (primer sequence 1)
SEQ ID NO: 2: PCR primer for preparing TCRVα14 Southern probe (primer sequence 2)
SEQ ID NO: 3: PCR primer for preparing TCRVβ Southern probe (primer sequence 3)
SEQ ID NO: 4: PCR primer for preparing TCRVβ Southern probe (primer sequence 4)
SEQ ID NO: 5: PCR primer for detecting TCRVα14-Jα281 (primer sequence 5) SEQ ID NO: 6: PCR primer for detecting TCRVα14-Jα281 (primer sequence 6) SEQ ID NO: 7: PCR primer for detecting TCRVβ2 (primer sequence 7)
SEQ ID NO: 8: PCR primer for detecting TCRVβ3 (primer sequence 8)
SEQ ID NO: 9: PCR primer for detecting TCRVβ4 (primer sequence 9)
SEQ ID NO: 10: PCR primer for detecting TCRVβ5 (primer sequence 10)
SEQ ID NO: 11: PCR primer for detecting TCRVβ6 (primer sequence 11)
SEQ ID NO: 12: PCR primer for detecting TCRVβ7 (primer sequence 12)
SEQ ID NO: 13: PCR primer for detecting TCRVβ8 (primer sequence 13)
SEQ ID NO: 14: PCR primer for detecting TCRVβ11 (primer sequence 14)
SEQ ID NO: 15: PCR primer for detecting TCRVβ12 (primer sequence 15)
SEQ ID NO: 16: PCR primer for detecting TCRVβ (primer sequence 16)
SEQ ID NO: 17: PCR primer for detecting TCRVβ (primer sequence 17)

It is a schematic diagram which shows the outline | summary of creation of a NKT cell-derived cloned mouse and establishment of an ES cell line. FIG. 4 is a schematic diagram of a TCRVα chain locus and a schematic diagram showing the positions of primers. It is the schematic diagram which shows the position of the schematic diagram of a TCRV (beta) chain locus, and a primer. The figure which shows the result of the Southern blot analysis using the genomic DNA of NKT clone mouse # 1 (tail and placenta), # 2 (tail and placenta), # 3 (placenta), # 4 (deadborn, placenta) (electrophoresis photograph) ). A white arrowhead indicates a band in which gene rearrangement has occurred, and a black arrow indicates a band before gene rearrangement has occurred (germline configuration). It is the figure (electrophoresis photograph) which proved using the PCR method that TCRV (alpha) 14-J (alpha) 281 gene rearrangement is performed in the clone mouse | mouth. It is the figure which showed the TCRV (alpha) chain | strand base sequence in a NKT cell origin clone mouse | mouth. It is the figure which showed the TCRV (beta) chain | strand base sequence in the clone mouse | mouth derived from an NKT cell. It is the figure (electrophoresis photograph) which showed the result of the Southern blot analysis which used NKT cell-derived ES cell (lane 1-6) genomic DNA. The probe is the same as that used for clone mouse analysis. A 13 kb band shift was observed with the TCRVα14 probe (lanes 1 to 6), and the disappearance of the 2.5 kb band was observed in the ES cells in lanes 3 and 4. Further, when the TCRVβ probe was used, a band after gene rearrangement was observed. The open arrowhead indicates the band where gene rearrangement occurred. The black arrow indicates a band before gene rearrangement (germline configuration). It is the figure (electrophoresis photograph) which showed the result of the Southern blot analysis by F1 by crossing NKT clone mouse | mouth # 1 and ICR seed | species mouse | mouth. F1 which inherited the 8 kb band derived from cloned mouse-derived TCRVα14 derived from gene rearrangement is seen in male 1 and female 6 (upper panel, arrow). In addition, alleles containing TCRVβ derived from cloned mouse # 1 are separated into about 9 kb and 8 kb, respectively (see FIG. 4, black arrowheads). Each F1 inherits a 10.4 kb band (open arrowhead) derived from the mother mouse and a band of about 9 kb or 8 kb. It is the figure which showed the result of the FACS analysis in F1 by crossing NKT clone mouse | mouth # 1 and ICR seed | species. The upper row shows the proportion of TCRVβ8 in the cloned mouse offspring as the proportion of all TCRVβ positive cells. The lower panel shows the percentage of NKT cells in the cloned mouse offspring. Panel 1 is a control ICR species mouse (wild type), Panel 2 is a mouse that inherits TCRVβ8 after gene rearrangement in frame, and Panel 3 is a gene rearrangement of Vα14-Jα281. Each shows the results of a mouse that exits and inherits it. NKT cell groups were circled and expressed as a percentage (%) as the number of NKT cells relative to the total number of cells. It is a figure which shows the result of having analyzed the state of differentiation or survival in the in vitro culture (48 and 72 hours) of the reconstructed embryo obtained by transplanting NKT cell nucleus or T cell nucleus, and shows the results at 48 and 72 hours in Table 1. It is a graph (except for the birth of a mouse). The vertical axis shows the survival rate of cultured embryos over time, or the incidence of individuals when the reconstructed embryo is introduced into pseudopregnant mice (the birth rate of transplanted embryos). ^* P <0.0001; ^** p <1 × 10 ⁻²⁵ It is a figure which shows the result (TCRV (beta) vs TCRV (beta) 8) of the peripheral lymphocyte of a clone mouse | mouth (# 1, # 2). Serum was collected from mice at 0, 4, 12, and 24 hours after α-GC stimulation, and cytokines (IFN-γ, IL-2, GM-CSF, IL-4, IL− in the obtained serum) were obtained. 10, IL-5, TNF-α, IL-1β) levels were measured by ELISA. Measurements were made on 4 mice at each measurement. Each cytokine concentration is shown on the vertical axis, and measurement points are shown on the horizontal axis. Data were expressed as mean ± standard deviation. IL in the culture supernatant of a mixed culture of dendritic cells (DC) (5 × 10 ⁴ cells) previously subjected to a-GC treatment and spleen cells (SPC) (1 × 10 ⁶ cells) as response cells -4 and IFN-γ were measured. The vertical axis shows the concentration of each cytokine, and the horizontal axis shows the origin of spleen cells. Data were measured in triplicate and expressed as the mean ± standard deviation.

Claims (31)

A method for producing a cloned non-human mammal, comprising using a natural killer T cell of a mammal as a donor cell. A method for producing a cloned non-human mammal, comprising introducing a nucleus of a mammalian natural killer T cell into an enucleated oocyte of a mammal. A clone non-human mammal comprising introducing a nucleus derived from a mammalian natural killer T cell into a enucleated oocyte of a mammal to form a reconstructed embryo, and transferring the reconstructed embryo to a host mammal How to create animals. The method for producing a cloned non-human mammal according to any one of claims 1 to 3, wherein the natural killer T cell has been genetically manipulated to express a desired character. The method for producing a cloned non-human mammal according to any one of claims 1 to 4, wherein the natural killer T cell and the enucleated oocyte are derived from the same mammal. The method for producing a cloned non-human mammal according to any one of claims 1 to 4, wherein the natural killer T cell and the enucleated oocyte are derived from different mammals. The clonal non-human of any one of claims 1 to 6, wherein the natural killer T cell is collected from a tissue selected from the group consisting of umbilical cord blood, peripheral blood, liver, bone marrow, spleen, and thymus. How to create a mammal. A cloned non-human mammal produced by the method according to any one of claims 1 to 7. A fetus of a cloned non-human mammal obtained by the method according to any one of claims 1 to 7. 9. A progeny of a cloned non-human mammal according to claim 8. The progeny of a clonal non-human mammal according to claim 10, characterized in that the TCRV β chain expressed on T cells is substantially composed of a single TCRV β chain repertoire. 12. The progeny of a cloned non-human mammal according to claim 11, wherein the single TCRV β chain repertoire is identical to the TCRV β chain repertoire of NKT cells used as donor cells. 11. The offspring of a cloned non-human mammal according to claim 10, characterized in that it has an allele containing Vα14-Jα281, a TCRα chain that has undergone gene rearrangement, and the number of NKT cells is increased. A cell, tissue, organ or product obtained from the cloned non-human mammal according to claim 8. A cell, tissue, organ or product obtained from the fetus of a cloned non-human mammal according to claim 9. A cell, tissue, organ or product obtained from the offspring of a cloned non-human mammal according to claim 10. 17. A cell, tissue, organ or product according to any one of claims 14 to 16, characterized in that it is immunologically identical to a donor cell. The cell, tissue, organ or product according to any one of claims 14 to 17, which is used for treatment, organ transplantation and / or cell transplantation. A method for producing a cloned mammalian embryo, characterized in that a mammalian natural killer T cell is used as a donor cell. A method for producing a cloned mammalian embryo, comprising introducing a nucleus of a mammalian natural killer T cell into a enucleated oocyte of a mammal. The method for producing a cloned mammalian embryo according to claim 19 or 20, wherein the natural killer T cell has been genetically manipulated to express a desired character. The method for producing a cloned mammalian embryo according to any one of claims 19 to 21, wherein the natural killer T cell and the enucleated oocyte are derived from the same species of mammal. The method for producing a cloned mammalian embryo according to any one of claims 19 to 21, wherein the natural killer T cell and the enucleated oocyte are derived from different mammals. The cloned mammal according to any one of claims 19 to 23, wherein the natural killer T cells are collected from a tissue selected from the group consisting of umbilical cord blood, peripheral blood, liver, bone marrow, spleen, and thymus. How to make an embryo. A cloned mammalian embryo produced by the method according to any one of claims 19 to 24. 26. Culturing the cloned mammalian embryo of claim 25 to the blastocyst stage or pre-blastocyst stage, and isolating embryonic stem cells from the resulting inner cell mass of the blastocyst stage or pre-blastocyst stage A method for producing a cloned mammalian embryonic stem cell. A cloned mammalian embryonic stem cell obtained by the method of claim 26. A method for producing a differentiated cell, tissue, organ or product of a cloned mammal, comprising culturing and inducing differentiation of the cloned mammalian embryonic stem cell according to claim 27 under conditions capable of inducing desired cell differentiation. A differentiated cell, tissue, organ or product of a cloned mammal obtained by the method of claim 28. 30. A differentiated cell, tissue, organ or product according to claim 29, characterized in that it is immunologically identical to a donor cell. The differentiated cell, tissue, organ or product according to claim 29 or 30, which is used for treatment, organ transplantation and / or cell transplantation.